Abstract The four Sprouty proteins (Spry1, 2, 3, & 4) inhibit the biological actions of receptor tyrosine kinases (RTKs) and thereby regulate the development of various organs. Because of their role as inhibitors RTK- mediated cell proliferation and migration, decrease in Spry proteins, especially Spry2 has been implicated in a number of cancers. Thus, it is now well established that Spry2 protein levels are decreased in breast, lung, prostate and hepatocellular carcinomas (HCC). The co-PI of this proposal has shown that in patient-derived HCC tumors, marked decreases in Spry2 levels correlated with poor patient outcome. Moreover, in a subset of these HCCs, the decreased Spry2 levels inversely correlated with increased levels of Nedd4-1, which the PI's lab has shown is one of the E3 ubiquitin ligases that ubiquitylates Spry2 targeting it for degradation. The PI's lab has also found that Spry2 protein levels can be regulated by prolyl hydroxylation and von Hippel Lindau protein (pVHL)- associated E3 ligase. Others as well as we show that pVHL protein levels are elevated in subsets of HCCs. Additional findings from the PI's lab show that Spry2 is also SUMOylated and this post-translational modification increases its cellular content. Given these findings, the central hypothesis that this proposal will test is that Spry2 content in HCCs is regulated by Nedd4-1- and/or pVHL- mediated ubiquitylation and degradation or via alterations in its SUMOylation and that decreased Spry2 content in HCC elevates RTK signaling, thereby augmenting tumor formation and growth. To address this hypothesis, we will determine whether disrupting the endogenous Spry2/Nedd4-1 interactions enhance Spry2 content and, therefore its ability to inhibit migration and proliferation of HCC cells and HCC formation in mice. We will also determine whether interfering with Spry2 interactions with Spry2 and pVHL enhances its cellular content and also augments its ability to suppress HCC formation. Finally, we will elucidate the mechanisms by which Spry2 is SUMOylated, determine whether SUMOylation stabilizes Spry2 and regulates its biological actions in HCC cells, and also determine the status of Spry2 SUMOylation in HCC tumors. Our studies will not only identify novel mechanisms by which Spry2 levels are regulated but also provide insights into newer possibilities of therapeutic strategies for HCCs. Moreover, since Spry2 protein levels are decreased in breast, prostate, and lung cancer that afflict male and female Veterans, our studies will also have a broader impact in these diseases.